Inflammation Research

, Volume 66, Issue 2, pp 187–196 | Cite as

Demethyleneberberine alleviates inflammatory bowel disease in mice through regulating NF-κB signaling and T-helper cell homeostasis

  • Ying-Ying Chen
  • Rui-Yan Li
  • Mei-Jing Shi
  • Ya-Xing Zhao
  • Yan Yan
  • Xin-Xin Xu
  • Miao Zhang
  • Xiao-Tong Zhao
  • Yu-Bin ZhangEmail author
Original Research Paper



The activation of NF-κB signaling and unbalance of T-helper (Th) cells have been reported to play a key role in the pathogenesis of colitis. Cortex Phellodendri Chinensis (CPC) is commonly used to treat inflammation and diarrhea. Demethyleneberberine (DMB), a component of CPC, was reported to treat alcoholic liver disease as a novel natural mitochondria-targeted antioxidant in our previous study. In this study, we investigated whether DMB could protect against dextran sulfate sodium (DSS)-induced inflammatory colitis in mice by regulation of NF-κB pathway and Th cells homeostatis.


Inflammatory colitis mice were induced by 3% DSS, and DMB were orally administered on the doses of 150 and 300 mg/kg. In vitro, DMB (10, 20, 40 μM) and N-acetyl cysteine (NAC, 5 mM) were co-cultured with RAW264.7 for 2 h prior to lipopolysaccharide (LPS) stimulation, and splenocytes from the mice were cultured ex vivo for 48 h for immune response test.


In vivo, DMB significantly alleviated the weight loss and diminished myeloperoxidase (MPO) activity, while significantly reduced the production of pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and inhibited the activation of NF-κB signaling pathway. Furthermore, DMB decreased interferon (IFN)-γ, increased IL-4 concentration in the mice splenocytes and the ratio of IgG1/IgG2a in the serum. In vitro, ROS production and pro-inflammation cytokines were markedly inhibited by DMB in RAW264.7 cell.


Our findings revealed that DMB alleviated mice colitis and inhibited the inflammatory responses by inhibiting NF-κB pathway and regulating the balance of Th cells.


DMB Colitis NF-κB Th cell 



Carboxymethylcellulose sodium


Cortex Phellodendri Chinensis


Disease activity index






Hematoxylin and eosin


Inflammatory bowel diseases












N-acetyl cysteine


Reactive oxygen species


T helper


Th cell type 1


Th cell type 2


Tumor necrosis factor-α


Ulcerative colitis



This work was supported by National Natural Science Foundation of China Grant 81573484 to Y.B.Z., Opening Project of Shanghai Key Laboratory of Complex Prescription (Shanghai University of Traditional Chinese Medicine) (14DZ2271000), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and College Students Innovation Project for the R&D of Novel Drugs (J1030830).

Compliance with ethical standards

Conflict of interest

There are no competing interests to declare.

Supplementary material

11_2016_1005_MOESM1_ESM.doc (36 kb)
Supplementary material 1 (DOC 36 kb)
11_2016_1005_MOESM2_ESM.tif (1.3 mb)
Fig.S1 DMB was determined to be a strong antioxidant in vitro and vivo. (a) Results of ROS fluorescent staining with DCFH-DA with the flow cytometry. (b) Flow cytometric histograms of A. (c) T-AOC of DMB and BBR. ***, ###< 0.001. * p versus control group and # p versus LPS group. (TIFF 1300 kb)
11_2016_1005_MOESM3_ESM.tif (252 kb)
Fig.S2 The total p-p65 expression in RAW264.7 cell with the stimulation of LPS at different time. (TIFF 252 kb)
11_2016_1005_MOESM4_ESM.tif (1.1 mb)
Fig.S3 The time course of mRNA expression in RAW264.7 cell stimulated with 100 ng/ml or 500 ng/ml LPS for 6 h or 12 h. (a) The mRNA expression of IL-1β and TNF-α in RAW264.7 cell stimulated with 100 ng/ml or 500 ng/ml LPS for 6 h or 12 h. (TIFF 1138 kb)
11_2016_1005_MOESM5_ESM.tif (1 mb)
Fig.S4 The effect of DMB on the expression of p65 in the nucleus of RAW264.7 cell. (a) The western blotting result of p65 in the nucleus of RAW264.7 cell (b) The relative densities of western blotting band were compared with the Histon H3 band in each group. Values were shown as the means ± SEM of five independent tests. **<0.01 and ***, ###< 0.001. * p versus Control group and # p versus LPS group. (TIFF 1049 kb)
11_2016_1005_MOESM6_ESM.tif (1.1 mb)
Fig.S5 The HPLC analysis of DMB which move into the RAW264.7 cells. (a) The HPLC analysis of DMB standard and methanol solvent. (b) The HPLC analysis of control group in collected RAW264.7 cell which cultured without DMB. (c) The HPLC analysis of DMB and the third time washing PBS in collected RAW264.7 cell which cultured with DMB for 2 h at 2 mM concentration. (d-f) The HPLC analysis of DMB and the third time washing PBS in collected RAW264.7cell which cultured with DMB for 0.5, 1h and 2h at 2mM concentration (TIFF 1085 kb)
11_2016_1005_MOESM7_ESM.tif (744 kb)
Fig.S6 Effect of DMB on the expression of TLR4 in mice colon. (a) The protein expressions of TLR4 were examined by Western blotting. (b) The relative densities of western blotting band were compared with the β-actin band in each group. *<0.05 and ***, ###< 0.001. * p versus Control group and # p versus DSS group. (TIFF 743 kb)
11_2016_1005_MOESM8_ESM.tif (608 kb)
Fig.S7 The distribution of DMB in the rat tissues at different time. (a) Methanol HPLC results. (b - c) The DMB distribution in lung and brain at the 5 min. (d) The HPLC results of DMB distribution in the liver at the 30 min. (TIFF 608 kb)
11_2016_1005_MOESM9_ESM.doc (28 kb)
Supplementary material 9 (DOC 28 kb)
11_2016_1005_MOESM10_ESM.doc (28 kb)
Supplementary material 10 (DOC 28 kb)
11_2016_1005_MOESM11_ESM.doc (28 kb)
Supplementary material 11 (DOC 28 kb)
11_2016_1005_MOESM12_ESM.doc (30 kb)
Supplementary material 12 (DOC 30 kb)


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Copyright information

© Springer International Publishing 2016

Authors and Affiliations

  • Ying-Ying Chen
    • 1
    • 2
  • Rui-Yan Li
    • 1
  • Mei-Jing Shi
    • 1
  • Ya-Xing Zhao
    • 1
  • Yan Yan
    • 1
  • Xin-Xin Xu
    • 1
  • Miao Zhang
    • 1
  • Xiao-Tong Zhao
    • 1
  • Yu-Bin Zhang
    • 1
    Email author
  1. 1.State Key Laboratory of Natural Medicines, Department of BiochemistryChina Pharmaceutical UniversityNanjingChina
  2. 2.Department of Neurobiology and AnatomyNortheast Ohio Medical UniversityRootstownUSA

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